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Liver Receptor Homolog-1 Regulates the Expression of Steroidogenic Acute Regulatory Protein in Human Granulosa Cells

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9032

Address all correspondence and requests for reprints to: George R. Attia, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5322 Harry Hines Boulevard, Dallas, Texas 75390-9032. E-mail: george.attia{at}utsouthwestern.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Steroidogenic acute regulatory protein (StAR) plays a critical role in the initial step of steroid hormone synthesis. In the present study, we investigated the role of liver receptor homolog-1 (LRH-1) and dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome, gene 1 (DAX-1) in the regulation of StAR gene expression in human granulosa cell tumor cells. We also examined the effect of protein kinase A (PKA) signaling pathway on the expression of StAR in the presence of LRH-1 and DAX-1. Cell transfection, mutation analysis, and EMSA were performed. LRH-1 significantly induced StAR promoter activity in a concentration-dependent manner. This induction was further augmented in the presence of PKA agonist. Using deletion analysis, we demonstrated LRH-1 binding site at –105/–95. Mutation of this site resulted in a significant decrease in the StAR promoter activity. Using EMSA, the ability of this cis-element to bind LRH-1 was confirmed. DAX-1 inhibited LRH-1-stimulated StAR promoter activity in a concentration-dependent manner. This inhibition was also maintained in the presence of PKA stimulation. Our results demonstrated that LRH-1 plays a critical role in the induction of StAR gene expression. We hypothesize that LRH-1 could be the major transcription factor responsible for the rapid and significant increase in ovarian StAR gene expression after ovulation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
STEROIDOGENIC ACUTE REGULATORY protein (StAR) plays a critical role in the initial step of steroid hormone synthesis. It regulates the rate-limiting step in steroidogenesis by facilitating the transport of cholesterol to the inner mitochondrial membrane (1, 2). StAR, which is primarily localized in the gonads and adrenals (3, 4, 5), is rapidly synthesized in response to tropic hormone (6) and cAMP stimulation (7, 9).

Steroidogenic factor-1 (SF-1) is a key regulator of endocrine function within the hypothalamus-pituitary-gonadal axis (10, 11). It regulates the expression of several steroidogenic enzymes that are critical for reproductive function (12, 13, 14, 15, 16, 17, 18, 19). Using immunohistochemistry, SF-1 was found to be expressed in cyclic human ovary as well as in granulosa and theca interna of antral follicles (20). This pattern of expression suggests that SF-1 plays an important role in regulating human ovarian steroidogenesis.

Liver receptor homolog-1 (LRH-1) is another member of the orphan nuclear receptor family and binds to DNA as monomer. LRH-1 has an overall 60% amino acid similarity to SF-1 with virtually identical DNA binding domain. Initially, it was thought that LRH-1 is only expressed in tissues derived from the endoderm such as pancreas, liver, and intestine. More recently, LRH-1 was found in human ovaries and adrenals (21, 22), raising the possibility that LRH-1 could play a role in the regulation of steroidogenesis.

Dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome, gene 1 (DAX-1) is another orphan nuclear receptor (23, 24) that regulates the expression of steroidogenic enzymes. Several studies have demonstrated the colocalization of SF-1 and DAX-1 in several steroidogenic tissues. DAX-1 represses the SF-1-mediated transactivation of the StAR, side chain cleavage (CYP11A), and 3ß-hydroxysteroid dehydrogenase promoters (25, 26).

In this study, we examined the role of LRH-1 and DAX-1 in the regulation of StAR gene expression in human granulosa cells. We also examined the effect of protein kinase A (PKA) signaling pathways on the expression of StAR in the presence of LRH-1 and DAX-1.

	Materials and Methods

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Cell isolation and culture

Human granulosa cell tumor (HGCT) cells were isolated from patients undergoing surgical removal of ovarian granulosa cell tumor. The use of ovarian tissue was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas, Texas. To obtain granulosa cell tumor cells, a portion of the tumor was dispersed into single cells using constant gentle agitation in 0.025% trypsin in DMEM/F-12 medium (GIBCO BRL, Gaithersburg, MD) and antibiotics (37 C, 30 min x 8). After each time point, the cell suspension was collected, pooled, and 5% Nuserum (Becton Dickinson, Mansfield, MA) was added to inactivate the trypsin. HGCT cells were pelleted and resuspended in DMEM/F-12 medium. Cells were routinely subcultured using 0.05% trypsin and replated at a 1:3 split. All the experiments described in this study were conducted using cells in culture for 2–8 wk.

Human luteinized granulosa cells (HLGC)

HLGC were obtained by follicular aspiration from reproductive-age women (age 25–36 yr) undergoing oocyte retrieval for in vitro fertilization. Briefly, women were treated with GnRH agonist before and during follicular stimulation using recombinant human gonadotropin. After follicular aspiration, HLGC were isolated as previously described (27). Briefly, HLGC were washed twice with DMEM/F-12 medium (GIBCO BRL) and were incubated for 30 min at 37 C in DMEM/F-12 medium containing 0.1% hyaluronidase to disperse HLGC. The dispersed cells were resuspended in 20 ml medium and transferred to 50-ml tubes containing 3.5 ml Histopaque 1077 (Sigma Chemical Co., St. Louis, MO). HLGC were separated from red blood cells by centrifugation at 600 x g for 15 min. HLGC formed a thin layer between the Histopaque and the medium. HLGC were removed and washed three times using DMEM/F-12 medium containing 5% fetal bovine serum; 1% ITS Plus (Collaborative Research, Waltham, MA); 2% Ultroser G (IBF Biotechnics, Sepracor, Inc., Marlborough, MA); and antibiotics. The isolated cells were used to prepare nuclear extracts.

StAR-luciferase and expression vector constructs

A transient expression system using the luciferase reporter gene was used to characterize the human StAR promoter. A 1.3-kb fragment extending from position +39 to –1293 (18) was cloned into a pGL-3 Basic luciferase reporter plasmid (Promega Corp., Madison, WI). Three-deletion vectors were produced by appropriate enzyme digestion of pGL3-StAR vector: pGL3-StAR (–912 to +39) was produced by XhoI and PvuII digestion of –1.30-kb promoter fragment; pGL3-StAR (–165 to +39) was produced by XhoI and PvuII digestion of the –912 deletion; and pGL3-StAR (–62 to +39) was produced by BglII and HindIII digestion of PCR-amplified product of pGL3-StAR vector using the following primers: (5'-TGATGGCGTTTAGATCTCCTG-3' and 5'-CTTTATGTTTTTGGCGTCTTCCA-3').

Transient transfections and reporter assays

Twenty-four hours before transfection, HGCT cells were subcultured onto 12-well plates at a density of 80,000 cells/well. Fugene 6 (Roche, Indianapolis, IN) was used to transfect 0.5 µg of reporter plasmid and the indicated amounts of expression vectors. pcDNA3 empty vector was used to assure constant amounts of DNA per well for each transfection. After transfection, cells were incubated for 18 h before being treated with the agonists for 6 h in low-serum medium (DMEM/F-12 medium containing 0.1% Ultroser G), when indicated. Cells were assayed for reporter activity using the luciferase assay system (Promega).

Nuclear extract and EMSA

Nuclear extract was prepared from confluent HGCT cells and HLGC. Human LRH-1 was transcribed/translated using the TNT coupled reticulocyte lysate systems following the manufacturer’s instructions (Promega). Human StAR oligonucleotide probes were designed at position –111/–87 (5'-ATCGCTCTATCCTTGACCCCTTCC-3'). This oligonucleotide was annealed at 85 C for 5 min in annealing buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl₂, pH 8.0) and then slowly cooled to room temperature. The annealed oligonucleotide was end-labeled with 3000 Ci/mM [³²P]2'-dATP (Amersham Pharmacia Biotech, Piscataway, NJ) using T4 polynucleotide kinase (Invitrogen Corp., Carlsbad, CA) at 37 C for 30 min. Either nuclear extract (10 µg) or 3 µl of the in vitro LRH-1 protein was incubated with 30,000 cpm labeled probe at 37 C for 20 min in 30 µl binding buffer (20 mM HEPES, pH 8.0; 1 mM EDTA; 10% glycerol; 50 mM KCl; 2 µg of poly dI.dC/dI.dC; 1 mg/ml BSA; 10 mM dithiothreitol). The DNA-protein complexes were separated from free probe by electrophoresis using 4.5% polyacrylamide gel for 2 h at 150 V. The gel was dried and visualized after autoradiography at –70 C for 24 h.

Statistical analysis

Data were analyzed by ANOVA using StatPac software (StatPac, Inc., Minneapolis, MN).

	Results

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LRH-1 enhances StAR promoter activity in HGCT cells

To examine the role of LRH-1 in the regulation of StAR gene expression, HGCT cells were cotransfected with StAR promoter construct alone and with increasing concentrations of LRH-1 or SF-1 expression vectors. Maximal stimulation of reporter activity was observed using 0.5 µg/well for both vectors. LRH-1 increased StAR gene expression in a concentration-dependent manner (Fig. 1). LRH-1 cotransfection, however, was more effective (>15-fold) in the induction of the StAR reporter construct than was SF-1 (<8-fold) (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Effect of LRH-1 on the StAR promoter activity. HGCT cells were cotransfected with luciferase reporter construct containing StAR promoter (0.5 µg/well) and LRH-1 expression vector (0.01–0.5 µg/well). After recovery for 18 h, cells were lysed and assayed for luciferase activity. LRH-1 induced StAR promoter activity in a dose-dependent fashion and was significantly different from basal (*, P < 0.05; **, P < 0.001). Data are expressed as a percentage of the basal (StAR promoter construct plus empty pcDNA3.1 vector), which is set at 100%, and graphed as the mean ± SE values for the relative luciferase activity of pooled data from three to four experiments.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Effect of LRH-1 or SF-1 on StAR promoter activity. HGCT cells were cotransfected with StAR promoter constructs (0.5 µg/well) and with empty pcDNA3.1, LRH-1 (0.5 µg/well), or SF-1 (0.5 µg/well) expression vector. After recovery for 18 h, cells were lysed and assayed for luciferase activity. LRH-1 and SF-1 significantly induced StAR promoter activity (16-fold, **, P < 0.001; and 8-fold, *, P < 0.05, respectively). Data are expressed as a percentage of the basal (StAR promoter construct plus empty pcDNA3.1 vector), which is set at 100%, and graphed as the mean ± SE values for the relative luciferase activity of pooled data from three to four experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Deletion analysis of StAR promoter construct. HGCT cells were cotransfected with sequentially deleted StAR promoter construct (0.5 µg/well) and LRH-1 expression vector (0.5 µg/well). After recovery for 18 h, cells were lysed, and luciferase assay was performed. LRH-1 increased StAR promoter activity by 15-fold compared with basal (*, P < 0.05). This activity was significantly diminished to basal activity using –65 constructs. Data are expressed as a percentage of the basal (StAR promoter construct plus empty pcDNA3.1 vector), which is set at 100%, and graphed as the mean ± SE values for the relative luciferase (Luc) activity of pooled data from three to four experiments.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Mutation analysis of StAR promoter construct. Site-directed mutation of StAR promoter was performed using PCR. HGCT cells were cotransfected with mutated StAR promoter construct (0.5 µg/well) and LRH-1 expression vector (0.5 µg/well). After recovery for 18 h, cells were lysed, and luciferase activity was assayed. LRH-1 significantly induced StAR promoter activity by 16-fold compared with basal (*, P < 0.001). This activity was significantly decreased using construct mutated (Mut) at position –95 compared with the wild-type (WT) (*, P < 0.001). Data are expressed as a percentage of the basal (StAR promoter construct plus empty pcDNA3.1 vector), which is set at 100%, and graphed as the mean ± SE values for the relative luciferase (Luc) activity of pooled data from three experiments.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effect of PKA agonists on the LRH-1-induced StAR promoter activity. StAR promoter construct (0.5 µg/well) was cotransfected with LRH-1 expression vector (0.5 µg/well) into HGCT cells. After recovery for 18 h, cells were treated with dbcAMP (100 µM) for 6 h. Cells were then lysed, and luciferase activity was assayed. Treatment with dbcAMP increased basal StAR promoter activity 2-fold (*, P < 0.05). LRH-1-induced StAR promoter activity was further augmented in the presence of dbcAMP treatment (*, P < 0.05). Data are expressed as a percentage of the basal (StAR promoter construct plus empty pcDNA3.1 vector), which is set at 100%, and graphed as the mean ± SE values for the relative luciferase activity of pooled data from three to four experiments.

Because DAX-1 is involved in SF-1-induced steroidogenic enzyme suppression, we examined the role of DAX-1 in the regulation of StAR gene expression in HGCT cells. HGCT were cotransfected with StAR promoter construct, LRH-1 expression vector, and increasing doses of DAX-1 expression vector. LRH-1-induced StAR promoter activity was suppressed by DAX-1 in a concentration-dependent manner (Fig. 6A). This suppression was maintained in the presence of PKA pathway agonist (dbcAMP) (Fig. 6B).

View larger version (16K): [in this window] [in a new window]   FIG. 6. Effect of DAX-1 on the LRH-1-induced StAR promoter activity. HGCT cells were cotransfected with StAR promoter construct (0.5 µg/well), LRH-1 (0.5 µg/well), and increasing concentrations (0.001–0.5 µg) of DAX-1 expression vector. In some experiments, after recovery for 18 h, cells were treated with dbcAMP (100 µM) for 6 h. DAX-1 inhibited LRH-1-induced-induced StAR activity in a dose-dependent fashion compared with LRH-1-only-induced StAR activity (*, P < 0.05; **, P < 0.001), with maximal inhibition obtained at concentration of 0.1 µg/well (A). This inhibition was maintained in the presence of PKA agonist dbcAMP (*, P < 0.001) (B). Data are expressed as a percentage of control (StAR promoter construct plus empty pcDNA3.1 vector), which is set at 100%, and graphed as the mean ± SE values for the relative luciferase activity of pooled data from three to four experiments.

To confirm that LRH-1 interacts directly with a known putative LRH-1 binding site, synthetic oligonucleotides encompassing the cis-elements (–111 to –87) were generated and used for EMSA. In vitro-transcribed LRH-1 proteins bound to the radiolabeled oligonucleotides and formed a specific protein/DNA complex that was completely displaced by the addition of 100-fold molar excess of nonradiolabeled oligonucleotides. In addition, when the radiolabeled oligonucleotide probe was incubated with HGCT and HLGC nuclear extracts, a similar protein/DNA complex was formed. This complex was displaced by excess nonradiolabeled oligonucleotides (Fig. 7).

View larger version (74K): [in this window] [in a new window]   FIG. 7. LRH-1 binds to putative cis-element in the StAR promoter in EMSA. A labeled oligonucleotide (–102/–89) was incubated with an increasing amount of HGCT cells nuclear extract (1–10 µg), HLGC nuclear extract (10 µg), and in vitro-transcribed LRH-1 [human LRH (hLRH)] protein (3 µl). The resulting protein/DNA complexes (shown by arrows) were separated from the free probe by electrophoresis. The intensity of complex 1 (C1) band increased as the concentration of nuclear extracts increased. This band was completely displaced by the addition of 100-fold molar excess of nonradiolabeled DNA (cold probe). The same result was obtained using both HLGC nuclear extract and in vitro-transcribed LRH-1 protein. Similar results were obtained in two additional independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Human granulosa cells, which contain low concentrations of StAR before the ovulatory surge of LH, acquire large amounts of StAR during luteinization (29). SF-1 is a transcriptional regulator of steroidogenic enzymes essential for reproductive function, including StAR. However, the expression of SF-1 was found to be down-regulated in ovarian cells after LH surge (30, 31). This postovulatory diversion in the expression of StAR and SF-1 raises questions regarding the role of SF-1 in corpus luteum steroidogenesis.

LRH-1 is another member of the orphan nuclear receptor family. Recently, using semiquantitative RT-PCR and real-time PCR, we have demonstrated a higher level of LRH-1 and a lower expression of SF-1 in human corpus luteum compared with mature ovarian follicle (32). Several studies have also demonstrated a significantly lower level of ovarian SF-1 expression after ovulation (33, 34). This differential expression of LRH-1 and SF-1 between mature ovarian follicles and corpus luteum, and the fact that both orphan nuclear receptors share the same putative binding sites, raises the possibility that LRH-1 rather than SF-1 could be the transcription factor essential for the postovulatory surge in ovarian steroidogenesis.

The difficulty in obtaining human granulosa cells in sufficient quantities has slowed progress in defining the mechanism regulating steroidogenesis. Our laboratory has isolated cells from ovarian tumors and placed them in monolayer cell culture. These HGCT cells have retained many of the characteristics of normal human granulosa cells maintained in primary culture. This cell model is able to reproduce many of the differentiated functions of granulosa cells. Specifically, they have maintained the production of progesterone and are able to convert androstenedione to estradiol. This cell model also responds to forskolin and dbcAMP by increasing production of progesterone and estradiol. In addition, HGCT cells have continued to express steroid-metabolizing enzymes that are under the control of cAMP. To validate the use of this cell model as representative of granulosa cells, we used nuclear extract from HLGC and compared its binding activity to that of HGCT model. As shown in Fig. 7, there is a similar binding pattern between these two cell models. Furthermore, the density of the bands obtained from these two nuclear extracts was similar. This result would argue for comparable expression of LRH-1 in both the HGCT model and the HLGC.

Previous studies have demonstrated two SF-1 binding sites in StAR gene promoter and the importance of these sites for maximal promoter activity and cAMP responsiveness (9, 18, 35). Using deletion analysis, we were able to demonstrate the significance of the –105/–95 site. Mutation of this site resulted in a significant loss of the StAR promoter activity. Using EMSA, we have also demonstrated the specificity of this binding element. The second identified SF-1 site (–926/–918) was not an effective LRH-1 binding element. This difference between LRH-1 and SF-1 binding activity may be due to the slight differences between these two nuclear receptors’ DNA binding specificities or due to differences in the cell models. There might also be tissue-specific differences in the orphan nuclear receptors’ expression and their mechanisms of control and cis-elements involved in the regulation of StAR gene expression.

It is unlikely that SF-1 and LRH-1 are redundant transcriptional regulators within the ovary. There appears to be differential regulation of gonadogenesis and gonadal steroidogenesis by SF-1 and LRH-1. The fact that SF-1 knockout mice fail to develop gonads suggests a lack of expression or regulatory function of LRH-1 in the developing gonad. The role of these orphan receptors in gonadogenesis and gonadal steroidogenesis will be further elucidated with the development of ovary-specific SF-1, LRH-1, and double-knockout animal models.

In steroidogenic tissues, DAX-1 blocks steroid biosynthesis by impairing the expression of StAR gene (26, 36, 37). DAX-1 colocalizes with SF-1 during mouse development (38) and inhibits SF-1-mediated transactivation of target genes. In the present study, DAX-1 inhibited LRH-1-stimulated StAR promoter activity in a dose-dependent manner. This inhibition was also maintained in the presence of PKA stimulation. We postulated that this inhibition may be mediated through direct interaction of LRH-1 with DAX-1, which is the same mechanism involved in the interaction between DAX-1 and SF-1 (39).

In conclusion, our data demonstrate a significant induction of human StAR promoter activity by LRH-1. We therefore suggest that LRH-1 rather than SF-1 could be the major transcriptional factor responsible for the rapid and significant surge in ovarian steroidogenesis after ovulation.

	Footnotes

 
Abbreviations: DAX-1, Dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome, gene 1; dbcAMP, dibutyryl cAMP; HGCT, human granulosa cell tumor; HLGC, human luteinized granulosa cell(s); LRH-1, liver receptor homolog-1; PKA, protein kinase A; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein.

Received September 12, 2003.

Accepted March 9, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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